Hematopoietic stem cells (HSCs) exist as rare populations within blood-forming tissues that both self-renew and generate all blood cell lineages for the life of the host. HSCs have been isolated to relative purity in humans and mice, leading to a general understanding of their cellular and functional properties. Relatively little is known, however, of the genetic program underlying the embryonic development and subsequent maintenance of HSC fate. The development of zebrafish as a model system has introduced unbiased genetic approaches, such as forward genetic screens and high-throughput expression screens, to the study of vertebrate blood formation. Recent transgenesis technology combined with the external fertilization and transparency of zebrafish embryos allows early events in HSC specification, maintenance, and differentiation to be easily observed. In this proposal, these advantages will be utilized to fate map the earliest embryonic blood-forming cells. This will address many long-standing questions in the development of the immune system, including whether mesodermat derivatives give rise to blood precursors through hemangioblastic intermediates, and whether primitive and definitive HSCs share common ancestry. In addition, the tools will be developed to prospectively isolate both embryonic and adult HSC populations. Following the generation of cDNA libraries from each subset, high-throughput expression screens will be performed to identify novel gene functions in vertebrate stem cells. The overall goal of this K01 application is to establish a cell biological context in which to study the genetics of HSCs in zebrafish. Novel genes that function within HSCs and their progeny can subsequently be examined for similar roles in mammalian systems. This award will provide the candidate a period of mentored research in the field of zebrafish blood development. Research collaborations, and resources will assist the candidate in the development of the skills and autonomy required to become a successful, independent investigator.